Methods for the diagnosis and treatment of critically ill patients with endothelin, endothelin agonists and adrenomedullin antagonists

ABSTRACT

The ratio of concentrations of pro-adrenomedullin (pro-ADM)/pro-endothelin (pro-END) immunoreactivity in body fluids of critically ill patients is used as for the diagnosis, course control and prognosis, including an assessment of the mortality risk, of severe life threatening diseases. Further, a treatment of critically ill patients having high levels of pro-ADM but insufficient levels of pro-END immunoreactivities with a medicament comprising vasoconstrictive endothelin or its precursors, and/or endothelin agonists or adrenomedullin antagonists is provided.

This application is a divisional of U.S. patent application Ser. No.12/095,675, filed May 30, 2008, which is a National Stage filing of PCTInternational Application No. PCT/EP2005/012844, filed Dec. 1, 2005 andpublished as WO 2007/062676 on Jun. 7, 2007.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Sep. 9, 2010, isnamed BOEH5D01.txt and is 7,065 bytes in size.

The present invention relates to novel diagnostic, and derivedtherapeutic, methods useful for the diagnosis and treatment of a groupof severe, life threatening diseases. The invention can also beconsidered as relating to the field of intensive care medicine.

More specially, the invention is based on the surprising finding thatthe physiological release of two different vasoactive types of peptides,namely of peptides of the adrenomedullin and endothelin families, forwhich it was already reported that they can be found elevated duringpotentially life threatening conditions as e.g. sepsis and other severeconditions, if considered in combination, provides new and highlyrelevant information on the actual status of a critically ill patientand his prognosis. A detected imbalance of the peptides of saidfamilies, at least if determined by means of suitable methods providinginformation on the release of said peptides rather than on their actuallevel in a body fluid, above certain critical threshold concentrationlevels in the circulation of the patient, enables a physician tointervene and to restore said balance so that the patient gets a chanceto recover from an acute crisis which otherwise would lead to his death.

The diseases for which the invention is contemplated as being of highdiagnostic and therapeutic value comprise sepsis, SIRS, septic shock,MOF (multiple organ failure), severe systemic or local infections,especially bacterial infections, peritonitis, pancreatitis, local and/orsystemic inflammation, meningitis, trauma, ruptured aortic aneurysm,intoxication, endotoxemia, anuria, renal insufficiency, arterialhypertension, pulmonary hypertension, artheriosclerosis, several typesof cancer, for example colorectal cancer, congestive heart failure,cardiovascular diseases, coronary artery disease, ischemia,antiarrhythmic effects, renal and/or heart failure, organ injury, anddiseases related to growth, and others.

The invention is based on the surprising finding that high levels ofvasoactive peptides of the endothelin family, which can be determined bymeasuring peptides indicating a high physiological release of the potentvasoconstrictors as endothelin-1, in critically ill patients with highlevels of adrenomedullin can exert a beneficial, potentially life savingeffect. In the scientific literature, in which occasionally speculationsabout an antagonistic behaviour of adrenomedullin and endothelinpeptides can be found, high concentrations of the vasoconstrictors ofthe endothelin family are constantly considered as dangerous andundesirable. High levels of adrenomedullin, on the contrary, areregarded as positive. An example for such speculations is found in thepublication of Takayuki Shindo et al., Hypotension and Resistance toLipopolysaccharide-Induced Shock in Transgenic Mice OverexpressingAdrenomedullin in Their Vasculature, In: Circulation. 2000;101:2309-2316.

The applicants have determined the formation or release of peptides ofthe adrenomedullin and endothelin families in critically ill patients,using new assays measuring prohormone (propeptide) immunreactivitieswhich provide information on the physiological release of both types ofpeptides rather than on the actual levels of the “mature” peptides inthe circulation, and they found that under certain circumstancesexplained in more detail below in the examples in connection with theresults of measurements of sera of critically ill patients, a dramaticdecrease of measurable endothelin formation, while at the same time thelevels of released adrenomedullin are critically high, precedes thedeath of such patients.

Based on said findings the applicants have defined a condition called“low endothelin syndrome” (in the following briefly LES) or “endothelindeficiency syndrome” which is characterized by a life threatening changein the release phase of endothelin (more specially of “endothelin-r” or“END-r” as it is defined below) in relation to the release ofadrenomedullin (more specially “adrenomedullin-r” or “ADM-r” as it isdefined below). Said syndrome is characterized by a high systemicconcentration, or a high release rate, of the vasodilator adrenomedullinand, concomitantly, by an insufficient release rate of thevasoconstrictor endothelin (END-r). The consequence of such mismatch isan uncontrollable vasodilation and an insufficient supply of one or moreorgans with blood, i.e. a single or multiple organ failure.

In the present application the suffix “-r”, used in terms as END-r orADM-r, stands for “released”. It means that the parameter in question isnot the actual, more or less momentary or transient measurable serum orplasma concentration of the biologically active biomolecule (maturepeptide) in question but rather reflects its cumulative release over alonger period of time. It can be directly measured e.g. by measuring apeptide fragment formed from the physiological precursor peptide(pre-pro- or pro-peptide or -hormone) concomitantly with the formationof the active biomolecule, which fragment, however, has a considerablylonger half life in the circulation (a considerably slower degradationor clearance rate) than the active biomolecule. Assays for measuringADM-r are described in applicants' European patent EP 1 488 209 B1“Bestimmung eines midregionalen Proadrenome-dullin-Teilpeptids inbiologischen Flüssigkeiten zu diagnostischen Zwecken, sowie Immunoassaysfür die Durchführung einer solchen Bestimmung”. Assays for measuringEND-r are described in applicants' European patent EP 1 564 558 B1“Verfahren zur Bestimmung der Bildung von Endothelinen zu Zwecken dermedizinischen Diagnostik, sowie Antikörper und Kits für die Durchführungeines solchen Verfahrens”. The contents of both patents are herebyexpressly included into the present application by reference. Examples 1to 6 of the present application can be considered as excerpts from bothpatents to provide in the present application an enabling disclosure.

Both the measurable adrenomedullin and endothelin formation or release(ADM-r an END-r) are part of the relevant combined measurementsaccording to the present invention. As relevant parameter a “ratio” ofboth can be defined. It is explained in more detail below.

The invention, according to one of its aspects, relates to the use ofthe “ratio” of ADM-r/END-r, in particular a ratio pro-adrenomedullin(pro-ADM)/pro-endothelin (pro-END) or fragments or combinations thereof,for the diagnosis of the low endothelin syndrome (LES) in critically illpatients affected by one of the diseases/conditions listed above. Morespecially the invention relates to the measurement of said ratio for thecourse control and prognosis of such disease, for the assessment of themortality risk alone and/or in combination with a respective computerprogram.

In one of its preferred embodiments the invention relates to furtherinclude clinical and/or other information for the evaluation of suchrisk.

LES may comprise but is not limited to the association of diseases likethose mentioned above.

ADM-r and END-r are normally measured by determining in suitable bodyfluids, especially in the circulation (serum, plasma, whole blood) arelatively persistent fragment of their physiological precursor peptides(pre-pro- or pro-peptides or -hormones). What is measured, therefore,can also be regarded as pro-peptide immunoreactivity (pro-hormoneimmunoreactivity). The terms pro-ADM and pro-END relate to suchmeasurable immunoreactivity and shall comprise also amino acid sequencesshowing at least 75% homology, preferred at least 80% homology, morepreferred at least 90% homology to pro-ADM or pro-END, their fragmentsor precursors.

The term diagnosis also comprises prognosis, early prognosis and coursecontrol.

The invention further relates to the use of endothelin forms havingvasoconstrictor activity, more particular of END-1 and/or big-END, aswell as optionally of END-agonists (molecules binding to the ENDreceptor and excerting there the same or similar effects as thevasoactive endothelin) and/or ADM-antagonists (molecules binding to theADM receptor and blocking there the effects as the vasoactiveadrenomedullin) for the preparation of a medicament for the treatment ofLES and/or diseases comprising those mentioned above. LES can beconsidered as an acute metabolic disorder involving an imbalance ofcertain potent effector molecules. By applying to a patient the activephysiological molecule for re-establishing the required balance, similarto the administration of insulin to a diabetes patient, detrimentaleffects of a lack of said active physiological molecule can beoutbalanced. The molecules involved in LES are adrenomedullin (ADM) andendothelin (END). In sepsis patients the concentrations of endothelinand adrenomedullin are increased above normal, depending on the severityof disease (FIGS. 1 and 2). In a very severe state of sepsis precedingdeath, according to the present invention it was surprisingly found thatthe concentration of endothelin decreases dramatically (FIGS. 3 and 4).This, in view of persisting high concentrations of ADM, consequentlylead to a dramatic decrease in blood pressure. That dramatic decrease inblood pressure caused by the lack of endothelin (LES) frequently leadsto the death of the patient as is disclosed in the present invention.

With respect to the published knowledge concerning adrenomedullin andendothelin, reference is made to the two earlier applications mentionedabove. In addition, reference also is made to applicants' earliestpatent application relevant for the determination of pro-ADM and pro-ENDin sepsis, published inter alia as WO 00/22439. The following summarizesthe most important facts.

Adrenomedullin (ADM) was first described in 1993 as a new hypotensivepeptide containing 52 amino acids that was isolated from aphenochromozytoma. In the same year the precursor proteinpre-pro-adrenomedullin (pre-pro-ADM) containing 185 amino acids wasdescribed. The precursor protein contains a signal sequence of 21 aminoacids at its N-terminus. ADM comprises the positions 95-146 of thepre-pro-ADM amino acid sequence and is a splice product thereof. Anothersplice product is PAMP, a 20 amino acid peptide spliced from positions22-41 that follow the 21 amino acids of the signal peptide. The termsADM or pro-ADM used in the present invention comprise ADM, itsprecursors and fragments thereof.

So far ADM and PAMP are known to influence blood pressure. ADM is apotent vasodilator, the hypotensive effect is supposed to be located inthe C-terminal part of ADM. Peptide sequences at the N-terminus on thecontrary show hypertensive effects. PAMP was also shown to have ahypotensive effect, even though the mechanism of action seems to bedifferent.

In connection with the discussion of therapeutical aspects of thepresent application, the term “endothelin” (END) generally comprisesendothelin forms with vasoconstrictor activity, especially endothelin-1(END-1) and its precursor big-endothelin, but may also comprise relatedmolecules as e.g. endothelin-2 (END-2) or endothelin-3 (END-3) as wellas their precursors.

In connection with analytical determinations, “endothelin” generallymeans END-r as defined above. It is, however, within the scope of theinvention to determine or monitor active forms of endothelin, i.e.especially END-1 or big-endothelin, because their decrease and finallydisappearance from the circulation also is directly indicative of theonset of a critical, life-threatening crisis. Absolute values formeasurable concentrations, and ratios of measurable concentrations, ofcourse, are influenced by e.g. the molecular species actually measured,by the units in which the measurements are expressed, and by thecalibration of the assays used. The absolute numerical values reportedin the present application are relevant for the assays designs describedherein only. Since they may have to be adapted if e.g. another analyteis measured or the particulars of its determination are changed, it isnot practical to express the results of the present invention inabsolute numerical values. However, by following the teachings of thepresent application, including the described scoring of the measuredresults, the skilled person can easily determine the numbers and ratiosrelevant for any modified determination method.

Endothelin-1 comprises 21 amino acids and is the most potentvasoconstrictor known. 3 isoforms of END are existing: endothelin-1(END-1), endothelin-2 (END-2) and endothelin-3 (END-3), END-1 is presentin the highest concentrations and is the most potent. Endothelin-1 issynthesized in the endothelial cells, in lung, heart, kidney and brain.The primary translation product of the human endothelin-1 gene ispre-proendoethelin-1, comprising 212 amino acids. In the secretorypathway the signal peptidase removes the short N-terminal signalsequence, comprising 17 amino acids. The resulting pro-endothelin issubsequently processed to a still biologically inactive big-endothelin,comprising 38 amino acids, by the protease furin, which can be found inthe circulation. The mature biologically active endothelin-1 is formedfrom big-endothelin by endothelin converting enzyme. Endothelin exertsits biological effect by binding to specific receptors that arelocalized on muscle cells, myocytes and fibroblasts. This binding causesthe efflux of calcium, activation of phopholipase C and inhibition ofNa/K ATPase. Besides the vasoconstrictive effect, endothelin alsoregulates growth.

The concentration of ADM in the blood circulation and other biologicalfluids is increased significantly in a variety of diseases likecongestive heart failure, myocardiac infarction, renal diseases, highblood pressure, diabetes mellitus, in the acute phase of shock, sepsis,septic shock, multiple organ failure. PAMP concentrations are alsoincreased in some of the above mentioned diseases/disorders, however theplasma level is decreased relatively to ADM. It is known that in sepsisand septic shock unusually high concentrations of ADM are observed. Thisfinding is related to the hemodynamic changes that are typical for thecourse of disease in patients with sepsis and other severe diseases likeSIRS.

Increased plasma concentrations of endothelin-1 and big-endothelin wereshown for several diseases comprising cardiovascular diseases comprisingpulmonary hypertension, artherosclerosis, congestive heart failure,cardiac infarction, sepsis, septic shock and cancer.

A major cause of mortality due to sepsis is the so-called multiple organfailure, which is caused by a systemic vasodilation, including adecrease of blood pressure and a loss of priority supply of singleorgans and tissues. Endothelin is thought to play a role in the degreeof severity of sepsis and to play a role in the mortality of sepsis.

Analytical/Diagnostic Aspects of the Invention

The present invention in one of its aspects relates to the diagnosis ofLES and/or associated diseases comprising those mentioned above. Thepresent invention further relates to the concomitant determination ofthe levels of END-r and ADM-r, more specially of pro-END and pro-ADMand/or their fragments. The combined measurement enables the calculationof a ratio and/or an algorithm and the use of such a derived ratioand/or algorithm for the diagnosis, prediction, prognosis of course ofseverity and the risk of mortality of LES and/or the above mentioneddiseases (FIGS. 1 and 2).

The invention is based on the surprising finding that both values ofpro-ADM and pro-END are increasing in patients during course of disease,wherein the values of pro-ADM are increasing more than those of pro-END,leading to an increase of the overall ratio pro-ADM/pro-END until oneday before exit.

More remarkably, however, the concentration of endothelin on the day ofdeath decreases dramatically. Consequently the ratio and/or algorithm ofpro-ADM/pro-END increases dramatically (FIGS. 3 and 4).

The determination of ADM, more specially ADM-r, as well as END, morespecially END-r, therefore provides information on the health state of acritically ill patient, especially with respect to the development of alife threatening crisis which requires an intervention. This isexplained more completely referring to the following analytical findingsand the results shown in Table 1 (see Example 8) and the Figures:

-   149 patients were included in a study, who were admitted to an    intensive care unit (ICU) and developed a sepsis during their stay    at ICU.

EDTA-plasma of the patients was gained at each day of their stay at ICU(3-29 days) . The plasma was stored at −20° C. until further use.

Pro-ADM and pro-END values were determined by using a coated tube assayas described in the earlier European patents of the applicant alreadymentioned above. For the assays, synthesized peptides were used toproduce antigens and injected into animals to raise antibodies againstthe different peptides as described earlier. In a preferred embodimentthe peptides were linked to the carrier protein keyhole limpethemocyanin using MBS (m-maleimidobenzoyl-N-hydroxysuccinimid ester) andused for the immunisation of sheep.

More particular, amino acid sequences of partial peptides 83-94[SSPDAARIRVKR=Sequence ID 2] and 69-86 [RPQDMKGASRS-PEDSSPD=Sequence ID3] of pre-pro-ADM 1-185 [Sequence ID 1] as well as partial peptide168-181 [RSSEEHLRQTRSET=Sequence ID 6] and 200-212[SRERYVTHNRAHW=Sequence ID 7] of pre-pro-END 1-212 [Sequence ID 5] weresynthesized. These sequences are comprised in the pre-pro-ADM 1-185 andpre-pro-END 1-212. An amino terminal cystein residue was added to eachpeptide. The peptides were conjugated to a hemocyanin and antibodieswere produced to the above mentioned peptides in sheep as describedearlier. Polyclonal antibodies were raised against the above mentionedpeptides. Antibodies were purified. In a preferred embodiment, this wasachieved by ligand specific affinity chromatography by coupling thepeptides via the amino terminal cystein residue to SulfoLink-Gel ofPierce (Boston, USA) according to the methods of Pierce. The antibodiesfurther were tagged with a marker to enable detection. The marker usedis preferably a luminescent marker and yet more preferred achemiluminescent marker as described earlier.

Methods for the detection of binding of the antibody to the respectivemolecule are known by the person skilled in the art comprising sandwichimmuno assays, radio immuno assays, ELISA or point of care tests.Preferably, according to the invention the binding of the antibody tothe target is detected by chemiluminescence.

The detection and the determination of the concentration of pro-ADM andpro-END can be conducted in various body fluids, tissues and otherbiomaterials but preferably is conducted in serum or plasma. It can takeplace in various states of all kinds of diseases as exemplified abovewhich may lead to LES, sepsis, septic shock, SIRS and related severeconditions.

It is part of the invention to use the correlation of ADM-r and END-r,preferably of precursor proteins with pro-ADM and pro-ENDimmunoreactivity, or, if feasible, also of the mature proteins like ENDand ADM in the state of disease/disorder in body fluids, tissues orother biomaterials, blood, plasma and liquor in particular.

The invention further uses the significant change of precursor proteinconcentrations in body fluids, tissue and other biomaterials in diseaseor disorder, in particular in a severe state of disease accompanied byLES as acute complication.

AMD-r, if measured as decribed herein, is present in plasma of healthyindividuals at concentrations of <1 nmol/l (median: 0,7). Criticallydiseased persons who survived a sepsis show a significantly increasedADM-r concentration (median 3,1). The median of dying patients issignificantly increased as compared to the group of surviving patients(median 11,8/17,1/17,7; 2 days before exit/one day before exit/on theday of death respectively, FIG. 1).

The concentration of END-r as a putative antagonist of ADM, if measuredas described herein, increases from a median of 0,035 nmol/l plasma to0,054 nmol/l in surviving patients. The concentration in dying patientsfurther increases to 0,096 and 0,133 nmol/l two days before exit and oneday before exit respectively. But surprisingly the concentration ofEND-r decreases dramatically on the day of death (median 0,064 nmol/l,FIG. 2).

These findings can be correlated with the prognosis of survival in asevere state of LES or other diseases comprising those mentioned aboveby determining the concentration of ADM-r, END-r and combinationsthereof and forming, as additional parameter, a ratio of the abovementioned proteins and/or an algorithm thereof.

Firstly, the concentration of ADM-r alone was used to determine theprognosis of survival (Table 1 and FIG. 1). Column 2 (specificity) showsthe number of surviving patients having a concentration of less than 17nmol/l serum/plasma of ADM-r, i.e. 117 of 126 patients have aconcentration of less than 17 nmol/l, 102 patients a concentration ofless than 8 nmol/l. Thus AMD-r shows a specificity of 93% for survivalat a cut off at 17 nmol/l and 81% at a cut off at 8 nmol/l.

The sensitivity of mortality is determined in the following way: 23patients died during their stay at ICU. At a cut off value of 17 nmol/lof ADM-r six patients (26%=sensitivity of mortality) showedconcentrations above 17 nmol/l two days before exit, 12 patients (52%)one day before exit and 13 patients (57%) on the day of death. At a cutoff value at 8 nmol/l, 19 (83%) of 23 patients showed concentrationsabove 8 nmol/l of ADM-r two days before exit, 21 patients (91%) one daybefore exit and 19 patients (83%) on the day of death (Table 1; seeExample 8).

Thus a cut-off value at 8 nmol/l of ADM-r (i.e. excluding the patientshaving a concentration below 8 nmol/l of ADM-r) includes most of thepatients who died during their stay at ICU.

Further, as has been found by the inventors, the ratio of ADM-r/END-raccording to the present invention forms an additional parameter of highvalidity in connection with the prognosis of septic patients in ICUs.

Surprisingly said ratio increases during the course of disease and evenmore surprisingly the ratio increases dramatically on the day of death.According to the results, the specificity and sensitivity of survivaland mortality respectively of LES and various other diseases comprisingthose mentioned above are disclosed, as well as the prognosis ofseverity of disease and mortality risk.

Measured ratios of AMD-r/END-r are shown in Table 1 and FIG. 3. TheADM-r/END-r ratio in healthy individuals has a maximum value of about 50(median 20). The median of surviving patients at ICU is increased to 56.Significant further increases can be observed two days (median 135) andone day (median 129) before exit and a surprisingly dramatic increase onthe day of death at a median of 336. The prognosis at a specificity of76% (Table 1, i.e. patients included having a concentration of ADM-r ofless than 43 nmol/l) is 91% two days before exit, 96% one day beforeexit and 87% on the day of death (Table 1 and FIG. 3). At a higherspecificity of 93%, (i.e. patients included having a concentration ofADM-r of less than 18 nmol/l), a prognosis is only possible on the dayof death having a sensitivity of 61% (Table 1).

It can surprisingly be deduced from the results that the threatening oflife in LES is only present at high ADM-concentrations, as a highpotential of vasodilation must be reached. High concentrations of ADMare only tolerated if they are outbalanced by the respective ENDconcentration.

For a further analysis of the results, the patients were furtherselected:

-   1. according to the AMD-r-concentration (>8 nmol/l) and-   2. according to the ratio of ADM-r/END-r (FIG. 4).

It was shown that a high portion of surviving patients with an ADM-rconcentration>8 nmol/l at one day of their stay at the ICU, N=24 had aratio of less than 90 (N=15) and thus their survival was assured.According to that assumption however also the dying patients would beincluded with a high probability 2 days before their death (sensitivity87% two days before exit/91% one day before exit/78% on the day ofdeath). Taking into account the sum of patients included in the study(126 surviving patients) the specificity surprisingly is as high as 93%.

The sequential analysis of ADM-r, END-r and the ratio and/or thealgorithm of ADM-r/END-r at an equally high specificity (93%)surprisingly shows a higher sensitivity for the prognosis (87%/91%/78%)than the analysis of ADM-r alone (26%/52%/57%) or ratio alone (0/0/61)(values 2 days before death/1 day before death/day of deathrespectively).

The invention, consequently, also relates to the detection, earlydetection, determination of severity, course control and prognosis ofthe diseases/disorders mentioned above using said ratio or thedetermined algorithm.

In this context the invention contemplates the use of a computer programto use the data received from the ratio for the detection, earlydetection, determination of severity, course control and prognosis ofLES and/or the diseases mentioned above.

For carrying out measurements as discussed above, assays for ADM-r andEND-r, as they are described in applicants' European patents mentionedabove, and the corresponding kits, can be used with advantage.

Therapeutical Aspects of the Invention

In contrast to the state of the art, the invention discloses thesurprising finding that elevated concentrations of endothelin(especially if determined as END-r) are beneficial for a patient havinghighly elevated concentrations of ADM-r as they are typically found inpatients in severe conditions as e.g. sepsis and the related diseasesmentioned above. In other words, the. results show that high levels ofEND-r enable a patient to tolerate high, otherwise deadly levels ofADM-r typical for conditions as sepsis so that death can be prevented.Shortly, high ADM levels apparently can be outbalanced by physiologicalEND levels of sufficient height.

A measurable decrease of END-r, or serum END, in such critically illpatient constitutes a clear clinical sign indicating a high risk ofdeath of patients having high levels of ADM-r. The observed sharpdecrease of measurable END-r and END respectively may be interpreted assign of an exhaustion of the cellular reservoirs of END precursors underthe physiological stress of a condition as sepsis, or of an inability ofthe physiological, molecular machinery producing END and its precursorsto keep up with the parallel continuing production of ADM and/or tooutbalance the continuing vasodilatory action of high ADMconcentrations.

According to a first therapeutical aspect the invention contemplates theadministration of vasoconstrictive endothelin forms, especially of END-1and/or big-END, to critically ill patients having high measured levelsof ADM-r to externally replenish the active END in such patients.“Vaso-constrictive endothelin forms” are intended to include directlyacting endothelin forms as END-1, as well as precursors as big-END whichas such are inactive but are readily converted to an active form whenthey are introduced into the circulation of a human patient.

The administration of endothelin, in clinical settings as e.g. in ICUs(intensive care units), can be preceded by an analysis of the actualconcentrations of ADM-r, END-r and a determination of the “ratio”discussed above. However, such preceding measurement and analysis mustnot be considered as an indispensable pre-requisite for theadministration of endothelin according to the present invention.

In health care settings, e.g. in an ICU, there may not be enough time towait for results of measurements of biomolecules as e.g. ADM-r and END-rbefore a decision regarding a therapeutic intervention can be made. Itis, therefore, within the scope of the present invention to administeractive endothelin to critically ill patients in response tocharacteristic changes of other physiological parameters which areindicative of the underlying low endothelin syndrome (LES). Suchparameters may be measurements of physiological variables as bloodpressure, body temperature, pulse frequency and similar parameters asthey are used to monitor the state of a septic patient. Knowing thebeneficial effect of an administration of endothelin, suchadministration can be effected on the basis of purely empirical clinicaldata.

It is, however, also within the scope of the present invention toadminister endothelin in response to a quantitative or semi-quantitativemeasurement of ADM-r alone, i.e. when the ADM level gets criticallyhigh. In view of the requirement to take a quick decision, it may bepreferable to conduct the determination of ADM-r in ICUs with POC(point-of care) measuring devices, e.g. devices of theimmunochromatographic type or devices with electrochemical probesquickly producing readings for the concentration of interest, to obtainan at least semiquantitative reading allowing an evaluation of theactual levels of ADM-r in a patient and to make a decision for theintravenous administration of endothelin. Suitable POC measuring devicesare well known to the skilled person. An example for a suitable designis the rapid test device PCT-Q® obtainable from B.R.A.H.M.SAktiengesellschaft for the rapid POC determination of procalcitonin(PCT) e.g. in a hospital setting.

It is further within the scope of the present invention to administerendothelin to patients which are continuously monitored for theconcentration(s) on one or more of the relevant biomolecules, or otherrelevant clinical parameters.

As stated earlier, in a therapeutical context “endothelin” meansendothelin forms with vasoconstrictor activity, especially endothelin-1(END-1) and its precursor big-endothelin, but the term may also extendto related molecules of similar activity, as e.g. endothelin-2 (END-2)or endothelin-3 (END-3) as well as their precursors.

The administration of the most active, directly acting END-1 at themoment is regarded as being preferred in critical care situations.Preferably such administration is an intravenous administration byinfusion or injection, using a liquid formulation (solution ordispersion) containing a suitable concentration of END-1 together withpharmaceutically acceptable carriers etc. Such liquid formulation(medicament) can contain all pharmaceutically acceptable and usefulexcipients, stabilisers and other ingredients suitable for such type ofadministration and for maintaining a stable condition within theadministered formulation for the time of infusion.

It is, further, within the scope of the invention to administer, orco-administer, slower acting forms of endothelins, for exampleadmixtures of quick acting and slower acting forms. Slower acting forms,e.g. in the form of big-END, may provide a more uniform pharmacokineticprofile and a longer lasting action of endothelins. Such forms may bemore suitable for injection, while directly acting forms are preferredfor administration by infusion.

It is further within the scope of the present invention to administeragonists of endothelin in place of, or together with, vasoconstrictiveendothelins to achieve special effects, e.g. a favourablepharmacokinetic profile, or to assist the patient in situations when themost critical acute phase is has been survived.

Further, since the life-threatening influence for the patient is not thelack of endothelin per se but the vasodilating action of highconcentrations of ADM (ADM-r), it is also within the scope of theinvention to use, in place of endothelin and/or endothelin agonists, orin combination therewith, adrenomedullin antagonists, i.e. moleculeswhich prevent or attenuate the vasodilating action of adrenomedulin,e.g. by blocking its relevant receptors, or substances preventing thebinding of adrenomedullin to its receptor (e.g. specific binders as e.g.antibodies binding to adrenomedullin and blocking its receptor bindingssites; “immunological neutralisation”). Such use, or combined use,including a subsequent or preceding separate use, may in certain casesbe desirable for example to improve the therapeutic success, or to avoidundesirable physiological stress or side effects.

The invention further discloses the use of endothelin forms withvasoconstrictor activity, more specially END-1 and/or big-END, as wellas END agonists, for the preparation of a medicament for the treatmentof LES and other conditions and diseases comprising those mentionedabove. In particular, endothelin-1, big-endothelin and/or any endothelin1 receptor agonists are used as active ingredients to preparemedicaments for administering them to a critically ill patient.Preferably the medicament is a liquid formulation for intravenousadministration by infusion, for example via a catheter, or by injection.

There do not seem to exist, in the available scientific literature,proposals to use vasoconstrictive endothelin for medical purposes. Theclaimed use, therefore, apparently can be considered as first medicaluse.

In medicaments as discussed above the mentioned active END may be used,as is customary in the art, in liquid formulations containing suitableconcentrations of e.g. appropriate carriers, stabilizers, nutrients,electrolytes, dissolved salts, osmotic agents for providing isotonicconditions, buffers and/or solvents and other adjuvants and excipientssuitable for administration in a patient.

DESCRIPTION OF THE FIGURES

FIG. 1 shows the concentration of ADM-r in plasma of 40 healthy controlindividuals, 126 surviving patients at ICU and 23 patients, two days andone day before death and on the day of death.

FIG. 2 shows the concentration of END-r in plasma of 40 healthy controlindividuals, 126 surviving patients at ICU and 23 patients, two days andone day before death and on the day of death.

FIG. 3 shows the ratio of ADM-r/END-r in plasma of 40 healthy controlindividuals, 126 surviving patients at ICU and 23 patients, two days andone day before death and on the day of death.

FIG. 4 shows the ratio of ADM-r/END-r including only patients having aADM-r concentration>8 nmol/l in plasma; 24 being surviving patients atICU and 20 further patients, two days and one day before death and onthe day of death.

EXAMPLE 1 Peptide Synthesis

An N-terminal cystein residue was added to synthesized Peptides of humanpro-ADM and pro-END. They were purified using mass spectrometry and thequality controlled using reversed phase HPLC and lyophiliysed inaliquots (Jerini AG, Berlin, Germany) according to standard proceduresknown to the person skilled in the art.

EXAMPLE 2 Conjugation and Immunization

Peptides of Sequence IDs 2, 3, 6 and 7 were conjugated to the carrierprotein KLH (keyhole limpet hemocyanin) by MBS(Maleimid-obenzoyl-N-hydroxysuccinimid ester) according to the protocolsfor “NHS-esters-maleimide crosslinkers” by PIERCE, Rockford, Ill., USA.Sheep were immunized receiving 100 μg of conjugate (.mu.g according tothe peptide content of the conjugate). Starting at month 4 afterimmunization every four weeks 700 ml of blood were withdrawn from everysheep and antiserum was gained by centrifugation. Conjugation,immunizations and production of antisera were done by MicroPharm,Carmerthenshire, UK.

EXAMPLE 3 Purification of Antibodies

The polyclonal antibodies from sheep were purified using ligand specificaffinity purification. For that step the Cys(0)-peptides were linked toSulfoLink-Gel supplied by Pierce (Boston, USA). The binding occurredaccording to the protocol of Pierce. 5 mg of peptide were added per 5 mlof gel.

In summary, columns were washed three times with 10 ml elution buffer(50 mM citric acid, pH 2.2) and binding buffer (100 mM sodium phosphate,0.1% Tween, pH 6.8). 100 ml of sheep antiserum were filtered using afilter diameter of 0.2 μm and added to the column material. The gel waseluted quantitatively with 10 ml binding buffer. The material wasincubated over night at room temperature by gentle rotation. Thematerial was transferred to empty columns (NAP 25, Pharmacia, emptied).The eluates were discarded. Subsequently the columns were washed with250 ml protein-free binding buffer (protein content of washedeluate<0.02 A280 nm). Elution buffer was added to the washed columns andfractions of 1 ml were collected. The protein content of each fractionwas determined by the BCA-method (according to the protocol of PIERCE,Rockford, Ill., USA). Fractions of a protein content >0.8 mg/ml werepooled.

EXAMPLE 4 Tagging

500 μl of affinity purified antibodies generated against the peptides ofSequence IDs 2, 3, 6 and 7 were rebuffered in 1 ml 100 mM potassiumphosphate buffer (pH 8.0) was re-buffered via a NAP-5 gel filtrationcolumn (Pharmacia) according to the protocol of Pharmacia.

For the tagging with a chemiluminescent marker 10 μl ofMA70-Akridinium-NHS-ester (1 mg/ml; Hoechst Behring) were added to 67 μlof antibody solution and incubated for 15 minutes at room temperature.The solution was rebuffered in 1 ml solvent A (50 mM potassiumphosphate, 100 mM NaCl, pH 7.4) in a NAP-5 gel filtration columnaccording to the protocols of Pharmacia, particles of low molecularweight were eluted. For elution of unbound labels a gel filtration HPLCwas done (Column: Waters Protein Pak SW300). The sample was added andchromatographes at a flow rate of 1 ml/minute in solvent A. The samplewas measured at wave length of 280 and 368 nm to determine the degree oftagging. The absorption ratio 368/280 nm had a peak of 1.0. Thefractions containing monomeric antibodies were collected (retention time8-10 minutes) and taken up in 3 ml 100 mM sodium phosphate, 150 mM NaCl,5% bovine serum albumin, 0.1% sodium azide, pH 7.4).

The tagged antibody was used in a sandwich assay as well as in differentassays like SPALT-assays.

EXAMPLE 5 Coupling

For a sandwich-assay the purified antibodies against the peptides ofSequence IDs 2, 3, 6 and 7 were immobilized on irradiated polystyroltubes (Greiner, Germany). For that procedure the antibody solutions werediluted to a protein concentration of 6.7 μg/ml with 5.0 mM Tris, 100 mMNaCl, pH 7.8. 300 .mu.l of diluted protein solution per tube werepipetted. These were incubated for 20 hours at room temperature, thesolution was removed. Then 4.2 ml of a 10 mM sodium phosphate, 2% KarionFP, 0.3% bovine serum albumin, pH 6.5 solution were added to each tube.After 20 hours the solution was removed and the tubes dried in a vacuumdrier. The procedure can also be done as inverse sandwich assayaccording to procedures known to a person skilled in the art.

EXAMPLE 6 Sandwich Immunoassay

The following assay buffer was used: 100 mM sodium phosphate, 150 mMNaCl, 5% bovine serum albumin, 0.1% unspecified sheep IgG, 0.1% sodiumazide, pH 7.4.

The protein content of EDTA-plasma of healthy individuals and patientsof various diseases/diseases mentioned above was determined.

In the tubes containing the immobilized antibody EDTA-plasma of patientswas pipetted (10 μl ADM and 50 μl END-r). 200 μl of PBS (phosphatebuffered saline), 0.1% bovine serum albumin were added to each tube andincubated at room temperature for 2 hours. After washing 3 times with 4ml PBS 200 μl acridiniumester-tagged polyclonal sheep antibody (affinitypurified, raised against the peptide 83-94 [Sequence ID 2] and 69-86[Sequence ID 3] of pre-pro-ADM 1-185 as well as partial peptide 168-181[RSSEEHLRQTRSET=Sequence ID 6] and 200-212 [SRERYVTHNRAHW=Sequence ID 7]of pre-pro-END 1-212, 20 ng each in PBS, 0.1% bovine serum albumin)respectively. After further incubation of 2 hours at room temperatureunbound tagged antibodies are removed by washing 5 times with 2 ml PBSeach. Tagged antibody bound to the tube was quantified by measuring theluminescence in a luminometer (Berthold LB 952T/16).

The individual values measured were quantified according to a usualcalibration curve that was established using defined dilutions ofsynthesized pre-pro-ADM 45-92 and pre-pro-END 169-212 as standardsolutions.

EXAMPLE 7 Measurements

Concentrations of ADM-r and END-r in Healthy Individuals and State ofDisease

149 patients were included in the study who were at the intensive careunit for different reasons and who developed a sepsis during their stayat the intensive care unit (criteria:www.talessin.de/scripte/medizin/sepsisl.html). Every day during theirstay at the intensive care unit (3-29 days) EDTA-plasma of the patientswas obtained and stored at −20° C. until further use.

ADM-r is present in plasma of healthy individuals at concentrations of<1 nmol/l (median: 0,7). Critically diseased persons who survived asepsis show a significantly increased ADM-r concentration (median 3,1).The median of dying patients is significantly increased as compared tothe group of surviving patients (median 11,8/17,1/17,7; 2 days beforeexit/one day before exit/on the day of death respectively, FIG. 1).

The concentration of END-r (pro-END) as a putative antagonist of ADMincreases from a median of 0,035 nmol/l plasma to 0,054 nmol/l insurviving patients. The concentration further increases to 0,096 and0,133 nmol/l two days before exit and one day before exit respectively.Surprisingly the concentration of END-r decreases dramatically on theday of death (median 0,064 nmol/l, FIG. 2).

EXAMPLE 8 Evaluations

ADM-r and the Prognosis for Severity of Disease/Disorder

The concentration of ADM-r was used to determine the prognosis ofsurvival (Table 1 and FIG. 1). Column 2 of Table 1 (specificity) showsthe number of surviving patients having a concentration of less than 17nmol/l plasma of ADM-r, i.e. 117 of 126 patients have a concentration ofless than 17 nmol/l , 102 patients a concentration of less than 8nmol/l. Thus ADM-r shows a specifity of 93% for survival at a cut off at17 nmol/l and 81% at a cut off at 8 nmol/l. The sensitivity of mortalityis determined in the following way: 23 patients died during their stayat ICU. At a cut off value of 17 nmol/l of ADM-r six patients(26%=sensitivity of mortality) showed concentrations above 17 nmol/l twodays before exit, 12 patients (52%) one day before exit and 13 patients(57%) on the day of death. At a cut off value at 8 nmol/l, 19 (83%) of23 patients showed concentrations above 8 nmol/l of ADM-r two daysbefore exit, 21 patients (91%) one day before exit and 19 patients (83%)on the day of death (Table 1).

Thus a cut-off value at 8 nmol/l of ADM-r, i.e. excluding the patientshaving a concentration below 8 nmol/l of ADM-r, includes most of thepatients who died during their stay at ICU.

TABLE 1 Specificity Sensitivity 2nd day- Last day before before day ofSurvivals death death death n = 126 n = 23 n = 23 n = 23 ADM-r Cut off117 6 12 13 17 nmol/l Cut off 102 19 21 19 8 nmol/l Ratio ADM-r/END-rCut off 215 117 0 0 14 Cut off 90 96 21 22 20 Ratio ADM-r/END-r ADM-r >8nmol/l Cut off 90 117 20 21 18 Specificity Sensitivity 2nd day Last daybefore before day of Survivals death death death ADM-r Cut off 93% 26%52% 57% 17 nmol/l Cut off 8 nmol/l 81% 83% 91% 83% Ratio ADM- r/END-rCut off 215 93% 0% 0% 61% Cut off 90 76% 91% 96% 87% Ratio ADM-r/END-rADM-r >8 nmol/l Cut off 90 93% 87% 91% 78%

EXAMPLE 9 Ratio

Ratio ADM-r/END-r

The ratio of ADM-r/END-r is shown in Table 1 and FIG. 3. The ADM-r/END-rratio in healthy individuals has a maximum value of about 50 (median20). The median of surviving patients at ICU is increased to 56.Significant further increases can be observed two days (median 135) andone day (median 129) before exit and a surprisingly dramatic increase onthe day of death at a median of 336. The prognosis at a specificity of76% (Table 1, i.e. patients included having a concentration of ADM-r ofless than 43 nmol/l is 91% two days before exit, 96% one day before exitand 87% on the day of death (Table 1 and FIG. 3). At a higherspecificity of 93%, i.e. patients included having a concentration ofADM-r of less than 18 nmol/l, a prognosis is only possible on the day ofdeath having a sensitivity of 61% (Table 1).

EXAMPLE 10

Ratio ADM-r/END-r Including Patients Having a Concentration of ADM-rAbove 8 nmol/l

Thus the patients were further selected according to theADM-concentration (>8 nmol/l) and according to the ratio of ADM-r/END-r(FIG. 4).

It was shown that a high portion of surviving patients with an ADMconcentration >8 nmol/l at one day of their stay at the ICU, N=24 had aratio of less than 90 (N=15) and thus their survival was assured.According to that assumption however also the dying patients would beincluded with a high probability 2 days before their death (sensitivity87% two days before exit/91% one day before exit/78% on the day ofdeath). Taking into account the sum of patients included in the study(126 surviving patients) the specificity surprisingly is as high as 93%.

Thus the sequential analysis of ADM-r, END-r and the ratio and oralgorithm of ADM-r/END-r at an equally high specificity (93%)surprisingly shows a higher sensitivity for the prognosis (87%/91%//78%)as the analysis of ADM (26%/52%/57%) or ratio (0/0/61) in their own(values 2 days before death/one day before death/day of deathrespectively).

The invention claimed is:
 1. A method of treating a critically ill humanpatient admitted to an intensive care unit (ICU) comprising: determiningthe concentration in nmol/l of a pro-adrenomedullin (pro-ADM) moleculeand/or one or more physiologically occurring fragments thereof using aplasma sample from the patient in an in vitro selective immunodiagnosticassay recognizing a physiologically occurring fragment that is not themature ADM, determining the concentration in nmol/l of a pro-endothelin(pro-END) molecule and/or one or more physiologically occurringfragments thereof using a plasma sample from the patient in an in vitroselective immunodiagnostic assay recognizing a physiologically occurringfragment that is not the mature END, wherein the immunodiagnostic assaysuse combinations of antibodies comprising antibodies produced againstpeptides having the amino acid sequences according to SEQ ID NO: 2 or 3for pro-ADM and SEQ ID NO: 6 or 7 for pro-END, calculating the ratio ofthe pro-ADM concentration to the pro-END concentration, and when theconcentration of pro-ADM is >8 nmol/l and the ratio of the pro-ADMconcentration to the pro-END concentration is above 90, administeringvasoactive endothelin in an amount sufficient to reduce the ratio ofpro-ADM concentration to the pro-END concentration to below
 90. 2. Themethod according to claim 1, wherein the patient is admitted to anintensive care unit with a diagnosis of one or more of sepsis, SIRS,septic shock, multiple organ failure, infections systemic or local,bacterial infections, heart disease, peritonitis, pancreatitis, localand/or systemic inflammation, meningitis, trauma, ruptured aorticaneurysm, intoxication, endotoxemia, anuria, renal insufficiency,arterial hypertension, pulmonary hypertension, atherosclerosis, cancer,congestive heart failure, cardiovascular disease, coronary arterydisease, ischemia, antiarrhythmic effects, cancer comprising colorectalcancer, renal and/or heart failure or organ injury.
 3. The methodaccording to claim 1 wherein at least one of the antibodies thatspecifically binds said physiologically occurring fragment of pro-ADM orpro-END is immobilized onto a solid phase and at least one secondantibody specifically binds another part of the same fragment and isused for detection of the fragment attached to the solid phase.
 4. Themethod according to claim 3 where the second antibody used for detectionis tagged with a luminescent marker.
 5. The method of claim 1 whereinthe vasoactive endothelin is administered by infusion or injection. 6.The method of claim 1 wherein the vasoactive endothelin is selected fromendothelin-1, big-endothelin, endothelin-2, endothelin-3 and anycombination thereof.
 7. The method of claim 1 wherein the vasoactiveendothelin is administered in a pharmaceutically acceptable liquidcarrier and one or more of stabilizers, nutrients, electrolytes,dissolved salts, osmotic agents for providing isotonic conditions,buffers and/or solvents and other adjuvants and excipients suitable forintravenous administration.